DESCRIPTION: The long term objective of this research program is to identify and characterize the various components of taste transduction pathways to understand how they function in the taste cell. Of particular importance are taste cell guanine nucleotide binding regulatory proteins (G proteins) and the seven transmembrane helix receptors that couple to them. G proteins regulate effector enzymes such as phosphodiesterase and phospholipase to effect taste cell changes in intracellular second messengers (e.g. cAMP, cGMP, IP3, Ca++). Gustducin is a taste specific G protein closely related to the transducins. Recently, it has been shown that rod transducin is also expressed in taste cells. These findings suggest that taste transduction may have some similarities to phototransduction. The specific goals of this proposal are the following: 1. To solubilize, reconstitute into lipid vesicles and physically characterize a recently identified apparent taste receptor that is responsive to denatonium. 2. To characterize the interaction of the presumptive denatoniu receptor with gustducin using peptide competition assays and directed mutagenesis of gustducin. 3. To chromatographically purify and molecularly clone this apparent receptor. 4. To characterize the gustducin-denatonium receptor interaction using behavioral and electrophysiological analysis of transgenic mice. Preliminary data demonstrate significant differences between gustducin knock out and wild type mice in their behavioral and electrophysiological responses to both sweet and bitter compounds. Additional preliminary data demonstrate that an 8.4 kb murine gustducin promoter driving expression of a gustducin cDNA transgene corrects the behavioral and electrophysiological deficits of gustducin knock out mice. 5. Transgenic mice will be made that express mutant forms of gustducin deficient in receptor interaction or with increased GDP/GTP exchange. 6. These transgenic animals will be tested behaviorally and electrophysiologically to determine if alterin the gustducin-receptor interaction or guanine nucleotide exchange affects bitter and sweet responses. Diverse methods will be required to address these goals: biochemical, transgenic, behavioral and electrophysiological techniques will be used. The results of these studies will provide significant new insights into the molecular mechanisms underlying bitter and sweet taste transduction. Gustatory and metabolic disorders such as malgeusia, dysgeusia and cachexia frequently occur in conjunction with several types of cancer. The knowledge gained from this proposal could further enhance our understanding of the molecular bases of taste disorders and may lead to effective intervention.